Babesia argentina: observations on the immunogenicity of the cryofibrinogen complex.

Rabbit antisera were prepared against the soluble and insoluble fractions of a cryofibrinogen complex that formed in plasma of cattle acutely infected with Babesia argentina. Analyses of the rabbit antisera indicated that the cryofibrinogen complex contained proteins from erythrocytes and parasites as well as fibrinogen and related proteins.     

Developing ways of reducing allograft immunogenicity.

Heavy alcohol consumption by the mother during pregnancy has long been suspected of being a risk factor for abnormalities in the fetus or infant. Only during the last decade have these assumptions been supported by scientific studies. A clustering of fetal defects observed in some cases has been labelled the fetal alcohol syndrome. The syndrome involves prenatal and postnatal growth retardation, central nervous system involvement and craniofacial abnormalities, some of which are characteristic of the syndrome. Fetal alcohol syndrome is relatively rare, affecting from 1 in 300 to 1 in 2000 infants; approximately 450 cases have been reported since the syndrome was identified. Despite this rarity, however, heavy alcohol consumption is an important risk factor during pregnancy. A review of the current literature indicates that in animals alcohol in high doses is embryotoxic and teratogenic, the heavy drinking is not uncommon before and during pregnancy and that the fetal alcohol syndrome and other effects on the fetus associated with alcohol abuse appear with significant frequency among mothers who drink heavily. Heavy alcohol consumption is a perinatal risk factor that not only can be detected by the physician, but also can be reduced in concerned, cooperative patients. Thus, awareness of this problem gives health care personnel an opportunity to help in the prevention of abnormal outcomes of pregnancy.     

BCG-CpG-DNA对重组HBsAg免疫原性的影响

为了研究BCG-CpG-DNA对重组HBsAg免疫原性的影响,了解其佐剂功能。采用不同剂量BCG-CpG-DNA与不同剂量重组(汉逊酵母)表达的HBsAg或Al-HBsAg混合免疫小鼠,与同剂量铝佐剂疫苗对比,以放射免疫法检测抗-HBs中和抗体水平和抗体持续时间,ELISA法检测抗体亚类。结果显示,BCG-CpG-DNA和HBsAg混合,抗-HBs的中和抗体水平达到或高于同剂量铝佐剂疫苗;BCG-CpG-DNA和Al-HBsAg混合,抗-HBs的中和抗体水平高于同剂量铝佐剂疫苗,并有统计学显著意义(P<0.05);添加BCG-CpG-DNA组抗体水平4周时增加不明显,到10周则显著地高于同剂量铝佐剂疫苗组,IgG2a抗体水平也高于相应的对照组。实验结果表明BCG-CpG-DNA对HBsAg有较好的免疫佐剂作用,并和AL佐剂有协同作用。     

The immunogenicity of intracerebral virus infection depends on anatomical site.

The brain parenchyma affords immune privilege to tissue grafts, but it is not known whether the same is true for intracerebral viral infections. Using stereotactically guided microinjection, we have confined infection with influenza virus A/NT/60/68 to either the brain parenchyma or the cerebrospinal fluid (CSF). A/NT/60/68 infection in the CSF elicited a comparable immune response to intranasal infection, with the production of antiviral serum antibody, priming of antiviral cytotoxic T-cell precursors, and an antiviral proliferative response in the draining lymph nodes. The response to virus in the CSF was detectable sooner after inoculation than the response to intranasal virus and also involved a prolonged production of virus-specific immunoglobulin A in the CSF. In contrast, there was no detectable immune response to virus infection in the brain parenchyma by any of the parameters measured for at least 10 days after inoculation. Over the next 80 days, 46% of the mice given parenchymal virus developed low-level immune responses that did not involve CSF antibody production, while the remaining 54% had no detectable response at any time. Thus, a virus infection confined to the parenchymal substance of the brain primed the immune system inefficiently or not at all.     

Effects of arildone on the immunogenicity of formalin-inactivated polioviruses

Concentrated and purified Sabin and virulent strains of poliovirus types 1, 2 and 3 were inactivated with formalin at 37 C. By addition of 5.4 microM arildone, an antiviral agent, to the virus suspension, the stability of D antigen increased in both Sabin and virulent strains of all types, especially in virulent type 1 Mahoney strain. The drug had neither any inhibitory nor enhancing effect on the formalin inactivation. When antibody response was compared in guinea pigs, Sabin strains inactivated in the absence of arildone were less immunogenic against homotypic virulent strains than inactivated vaccine prepared from virulent strains. On the other hand, Sabin strains inactivated in the presence of arildone were equally immunogenic. These results indicate that it is possible to prepare from Sabin strains a potent and safe inactivated vaccine having an immunogenicity comparable to that prepared from virulent strains.     

Scientific and regulatory considerations on the immunogenicity of biologics

Immune responses against non-vaccine biologics can affect their efficacy and safety, resulting in adverse events that could include administration reactions, hypersensitivity, deficiency syndromes and lack of a clinical response in treated patients. With the relatively recent development of numerous biologics, immunogenicity testing has become a key component in the demonstration of clinical safety and efficacy; in fact, it is highly unlikely that regulatory approval would be granted for a biologic without an assessment of its immunogenicity. However, recommendations from regulatory agencies regarding the requirements for when and how to carry out immunogenicity testing are dispersed among numerous guidance documents. To enable the evaluation of the effects of immunogenicity on safety and efficacy, the authors have consolidated recommendations from the regulatory guidelines, and present current approaches and future directions for the assessment of immunogenicity.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus.

A new approach to the preparation of antiviral vaccines relying on the inactivation of the virus particle by hydrostatic pressure is described. The enveloped virus vesicular stomatitis virus was utilized as a model; a pressure of 260 MPa applied for 12 h reduced infectivity by a factor of 10(4), and the antibodies against pressurized material were as effective as those against the intact virus when measured by their neutralization titer. Fluorescence measurements indicate that application of pressure results in perturbations of the particle interactions that permit binding of specific molecular probes. Electron microscopy showed that the membrane of the pressurized virus was partially preserved, presenting the spike pattern of the membrane G protein. Unlike the icosahedral viruses, dissociation into smaller particles was not observed, but a constant change in the morphology was the presence of a bulge in the surface of the pressurized virus, indicating a displacement of the capsid subunits, retained under the lipid and protein membrane.     

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.     

Immunoinformatics and the prediction of immunogenicity

Immunoinformatics is the application of informatics techniques to molecules of the immune system. One of the key goals of immunoinformatics is the development of computer aided vaccine design (CAVD), or computational vaccinology, and its application to the search for new vaccines. Key to solving this challenge is the prediction of immunogenicity, be that at the level of epitope, subunit vaccine or attenuated pathogen. This paper reviews the current state of play in the prediction of immunogenicity and focuses on well developed methods for the prediction of peptide binding affinity to major histocompatibility complexes, which are the necessary preliminary to the in silico identification of T cell epitopes.     

DTaP-Hib联合疫苗中Hib-TT免疫原性研究

考查DTaP-Hib联合疫苗中Hib-TT的免疫原性,对其剂量、免疫持久性和抗原相容性进行分析。将不同剂量的Hib-TT、DTaP-Hib联合疫苗分别免疫小鼠,设单价的Hib-TT结合疫苗为对照,末次免疫后1、2、4、6、8、10w分别采集血清测定血清中Hib多糖抗体滴度。结果显示,不同剂量的Hib-TT和DTaP疫苗联合后均具有较好的免疫原性,血清中Hib多糖抗体阳转率达100%,并具有剂量效应和较好的免疫持久性。2.5μg剂量Hib-TT的DTaP-Hib联合疫苗免疫小鼠后1~2w诱导产生的Hib多糖抗体水平显著性地低于单价Hib-TT(P<0.05),4~10w,二者的Hib多糖抗体水平无显著性差异(P>0.05)。5μg剂量Hib-TT的DTaP-Hib联合疫苗在免疫小鼠后1w诱导产生的Hib多糖抗体水平与单价2.5μg剂量Hib-TT无显著性差异(P>0.05),免后2~10w则显著性地高于单价2.5μg剂量Hib-TT(P<0.001)。Hib-TT和DTaP疫苗联合后,仍然具有较好的免疫原性、剂量效应和免疫持久性;其抗原性干扰只是暂时的。     

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.     

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens.

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.     

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.     

Elimination of the immunogenicity of therapeutic antibodies

The immunogenicity of therapeutic Abs limits their long-term use. The processes of complementarity-determining region grafting, resurfacing, and hyperchimerization diminish mAb immunogenicity by reducing the number of foreign residues. However, this does not prevent anti-idiotypic and anti-allotypic responses following repeated administration of cell-binding Abs. Classical studies have demonstrated that monomeric human IgG is profoundly tolerogenic in a number of species. If cell-binding Abs could be converted into monomeric non-cell-binding tolerogens, then it should be possible to pretolerize patients to the therapeutic cell-binding form. We demonstrate that non-cell-binding minimal mutants of the anti-CD52 Ab CAMPATH-1H lose immunogenicity and can tolerize to the "wild-type" Ab in CD52-expressing transgenic mice. This finding could have utility in the long-term administration of therapeutic proteins to humans.     

The effect of purification on the immunogenicity of tumor-specific transplantation antigens

Summary Immunization with the tumor-specific transplantation antigens (TSTA) of experimental, chemically induced sarcomas engenders specific host resistance to challenge with viable, homotypic neoplastic cells. The strength of tumor resistance depends upon the physical state of the TSTA used for immunization. Treatment with 10⁵–10⁶ irradiated tumor cells, a 2-log dose range, induces complete rejection of neoplastic challenges, while immunization within a 1-log dose range with crude 3 M KCl or with 2.5% butanol extracts containing TSTA evokes a weak state of resistance characterized by decreased outgrowth of tumor challenges, but not neoplastic regression. The reduced immunogenicity may be due to either contamination with substances that antagonize host resistance, for example by induction of suppressor cells, or an intrinsic limitation by virtue of the molecular properties of extracted compared with cell-surface TSTA. MCA-F and MCA-D, two noncross-reactive fibrosarcomas induced in C3H/HeJ mice with 3-methylcholanthrene, were employed to compare the relative immunogenic activity of intact tumor cells, 2.5% butanol extracts, and materials sequentially purified by preparative isoelectric focusing (pIEF), preparative isotachophoresis (pITP), and high performance gel permeation chromatography (HPGPC). Immunoprotective TSTA activity purified 50,000-fold by this protocol extended the effective dose range by four to five logs: 15 pg to 1.5 g MCA-F or 1 pg to 10 ng MCA-D antigen-induced specific host resistance. However, despite the appreciable purification of TSTA, immunization with extracted materials only delayed neoplastic outgrowth. They induced neither immediate rejection nor only temporary progression of transplanted tumor cells. Thus, purified TSTA preparations by themselves lack the immunogenic properties of intact cells that result in maximal induction of tumor resistance.